: Rationale: Carbapenem resistant Enterobacteriaceae (CRE), especially Klebsiella pneumoniae (KP) remain a significant public health threat. With a lack of treatment options, the polymyxins remain a mainstay of therapy. The rise of polymyxin resistant KP (PRKP) threatens these vital antibiotics. While modifications in the bacterial lipopolysaccharide (LPS) have proven the major mechanism of PR, a wide range of mutations in three two components systems (TCS), phoP/Q, crrA/B, pmrA/B, and mgrB, are thought to induce PR, but most have not been functionally validated. Additionally, little is known about how these mutations affect bacterial fitness and virulence, and if they can increase polymyxin minimum inhibitory concentration (MIC) independent of LPS modification. This mentored career development award aims to elucidate the downstream phenotypic effects of a broad selection of mutations in these genes. Candidate: As an infectious diseases clinician with a strong background in bacteriology and molecular biology, I am well suited to pursue translational research focusing on the determinants of antibiotic resistance. Further training in bacterial pathogenesis, anti-microbial resistance, bacterial genomics, and biostatistics will be crucial for the completion of the proposed research and advancement of my career. With primary mentor Dr. Anne-Catrin Uhlemann, I have assembled a multi-disciplinary team of experts to guide my training and research progress. My long-term goal is to become an independent NIH-funded researcher utilizing novel molecular biology techniques to characterize the determinants of bacterial antibiotic resistance and improve clinical practice. Environment: The Uhlemann laboratory at Columbia University Irving Medical Center has the microbiology, molecular biology and sequencing tools to complete the proposed research. The laboratory contains a large collection of CRE and PRKP clinical isolates that have undergone whole genome sequencing. Columbia has a long track record of supporting the career development of young investigators. Approach: Our central hypothesis is that the accumulation of multiple mutations in the PR cascade leads to rising MICs and changes in bacterial virulence through activation of unique cellular pathways. To elucidate the contribution of various mutations we will systematically insert these into two CRKP clinical isolates utilizing our CRISPR-Cas9 system (Aim 1). We will characterize how these changes affect MIC and LPS. In Aim 2 we will evaluate if PR can alter bacterial fitness and virulence through growth curves, co-incubation analyses, Galleria mellonella killing assays and a mouse pneumonia model. In Aim 3 we will utilize RNA-seq to characterize the differential phenotypes of the PR mutants by defining the cellular targets of the TCS. Through this we aim to identify novel pathways involved in PR and virulence and validate these targets through CRISPR mediated modification. In addition to elucidating how changes in the TCS induce PR and affect bacterial fitness, this work has the potential to identify novel pathways involved in PR and virulence. This would yield crucial information necessary for the diagnosis, treatment and prevention of PRKP infections.